Colloids and Surfaces B: Biointerfaces 115 (2014) 46–50

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Synthesis of highly fluorescent gold nanoclusters using egg white proteins Dickson Joseph a , Kurt E. Geckeler a,b,c,∗ a b c

Department of Nanobio Materials and Electronics, World-Class University (WCU) , South Korea School of Materials Science and Engineering, South Korea Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, South Korea

a r t i c l e

i n f o

Article history: Received 28 June 2013 Received in revised form 8 November 2013 Accepted 11 November 2013 Available online 20 November 2013 Keywords: Gold Nanocluster Photoluminescence Egg white Ovalbumin Protein

a b s t r a c t Gold nanoclusters (AuNCs) have gained interest during the recent years because of their low toxicity and finer size for the bioimaging and biolabeling applications in comparison to the semiconductor quantum dot analogues. Diverse materials such as sulfur compounds, peptides, dendrimers, proteins, etc., are exploited for the preparation of AuNCs. Henceforth, highly fluorescent, water-soluble, and few atomcontaining gold nanoclusters are created using a rapid, straightforward, and green method. In this regard for the first time chicken egg white (CEW), one of the most unique materials, is utilized in an aqueous solution under basic conditions at physiological temperature for the preparation of AuNCs. Tyrosine and tryptophan amino acid residues are responsible for the conversion of Au ions to Au0 under alkaline condtions. CEW contains four major proteins of which the main constituent protein, ovalbumin also leads to the formation of the AuNCs with a higher fluorescence emission compared to the CEW. The ratios between the different reaction partners are very crucial, along with temperature and time for the preparation of AuNCs with high photoluminescence emission. The limited vibrational motion of the proteins under alkaline condition and the bulkiness of the proteins help in the formation of AuNCs. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Gold nanoparticles (AuNPs) with a size below 1 nm are a collection of few tens of gold atoms, commonly known as gold nanoclusters (AuNCs). These ultrasmall gold nanoclusters have received attention for their unique role in bridging the “missing link” between the atomic and nanoparticle behavior [1–3]. A feature unique to AuNCs which distinguishes them from AuNPs is their highly fluorescent property [4]. Gold nanoparticles exhibit a sizetunable plasmon absorption band, when their conduction electrons in both the ground and excited states are confined to dimensions smaller than the electron mean free path (∼20 nm) [5], but the plasmon absorption disappears completely for nanoparticles less than 1 nm, where the Mie’s theory can no longer be applied [6–8]. Interestingly, metal nanoclusters confined to a second critical size scale regime, the Fermi wavelength of an electron (∼0.7 nm), results in molecule-like properties of discrete quantum confined electronic transitions [9–12] and size-tunable fluorescence [1,13]. The energy differences between the highest occupied and lowest unoccupied molecular orbitals are thought to scale as a function of the number

∗ Corresponding author at: Department of Nanobio Materials and Electronics, World-Class University (WCU), Gwangju 500-712, South Korea. E-mail address: [email protected] (K.E. Geckeler). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.017

of atoms within the clusters. Thus, differently sized nanoclusters exhibit different emissions. Fluorescence sensing and imaging techniques remain two of the primary methods for in vitro detection of molecules in solution and in vivo imaging of cells and cellular processes [14,15]. Relative to conventional organic dyes, semiconductor quantum dots show great promise in biolabeling due to their improved photophysical properties including size-tunable narrow emissions, large Stokes shifts, and minimal photobleaching. Unfortunately, semiconductor quantum dots are commonly synthesized using harsh conditions containing toxic metals, which are difficult to surface passivate, and have large physical size comparable to proteins, and tend to photoblink [16]. Each of these attributes limits the utility of semiconductor quantum dots for fluorescence imaging and sensing. In contrast to semiconductor quantum dots, AuNCs are highly attractive for bioimaging and biolabeling applications due to their low toxicity and ultra-fine size. The increasing use of metalcontaining compounds in therapy and diagnosis [17] has made metal nanoclusters an alternative as biomedical probes utilizing their luminescent properties. Gold nanoclusters protected by glutathione [18], tiopronin [19], meso-2,3-dimercaptosuccunic acid [20], and phenylethylthiolate [11] have been prepared via the chemical reduction method. However, the quantum yield of these gold nanoclusters reported is relatively low. The formation of quantum-confined, water-soluble,

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Scheme 1. A schematic representation of the experimental procedure for the formation of AuNCs using CEW proteins. The four major proteins in CEW: ovalbumin (OVA), ovotransferrin (OTF), ovomucoid (OMC), and lysozyme (LSZ) are displayed structurally, and the AuNC stabilized by the proteins. The color coding on the ribbon configuration, strand (green), monochrome (purple), secondary structure (blue) are for OVA, OTF, and LSZ, respectively, and the secondary structure (red) for OMC (For interpretation of the references to colour in this scheme legend, the reader is referred to the web version of this article).

and high quantum yield AuNCs embedded in poly(amidoamine) dendrimer has been reported, comprising repeatedly branched molecules with different generations [1,21]. The confined intraspace of the dendrimer restricts the growth of gold nanoclusters, but further purification through centrifugation is usually required to remove the large gold nanoparticles. Recently, Martinez and co-workers reported the nanoparticle-free synthesis of fluorescent gold nanoclusters via vitamin C [22]. Also, a ligand-induced poly(ethylenimine) etching process for the synthesis of highly fluorescent AuNCs with blue emission has been reported [23]. However, these synthetic procedures [1,22–24] require a fairly long reaction time. A novel “green” synthetic route for the preparation of redemitting gold nanoclusters using a protein was first reported by Xie and co-workers. Bovine serum albumin (BSA) was used as a template at physiological temperature to stabilize AuNCs within the protein molecules, which showed good biocompatibility as well as post-synthesis surface modification with functional ligands [25]. Following this report a bioactive protein was exploited towards the syntheis of AuNCs. The prepared insulin-AuNCs showed excellent biocompatibility and retained the insulin bioactivity. Inspite of the usage of the proteins as a green-chemical reducing and stabilizing agent in the above methods, the extraction of the proteins from their sources involves complex procedures [26]. Thus, the objective of this work was to utilize a simpler and cost-effective method to extract the proteins and utilize it for the syntheis of AuNCs. In this regard we have extracted the white portion of the chicken egg which mainly contains proteins and examined its experimental conditions to obtain highly fluorescent AuNCs. Chicken egg white (CEW) is a rich source of proteins, which is extremely cheap, easily available, and relatively well studied. They are globular proteins and most have isoelectric points in the acidic range [27]. On the basis of our understanding on protein chemistry [28] and gold nanoparticles [29], we were motivated to investigate CEW for the preparation of AuNCs. It has been well understood that in CEW, proteins are the principal components present making upto approximately 10.5% of its mass and the rest are water (88.5%), carbohydrates (0.5%), and other solutes [27]. Herein, we demonstrate a rapid, green, and straightforward synthesis of AuNCs using CEW under alkaline conditions at physiological temperature (37 ◦ C). One of the main advantages of using CEW over BSA is the method of the protein separation from its source. We separated the protein from CEW using a commonly used freeze-drying method. Since CEW contains 10.5% of proteins and the rest is water, it is easy to remove the water by freeze drying. While separating albumin from

bovine serum involves expensive methods that consists of multiple steps. 2. Experimental 2.1. Materials and characterization Potassium tetrachloroaurate (KAuCl4 ) was purchased from Aldrich. Sodium hydroxide was obtained from D.C. Fine Chemical Co., South Korea. All chemicals were used as received. Deionized (Milli-Q grade) water was used to prepare all solutions. The photoluminescence of the gold nanoclusters was measured using a Hitachi fluorescence spectrophotometer (F-7000). The UV–vis spectra were recorded on a PerkinElmer UV–vis spectrophotometer (Lambda 750). The Fourier Transform Infrared (FTIR) spectra were recorded as KBr pellets at room temperature under nitrogen atmosphere using a PerkinElmer FTIR spectrometer 2000. The TEM images were obtained with a JEOL JEM-2100 transmission electron microscope operating at 200 kV. The samples for TEM analysis were prepared by dropping the dispersions of nanoparticles on the carbon-coated copper grids and then drying them in an oven at 50 ◦ C for 12 h. 2.2. Synthesis of gold nanoclusters In a typical experiment, the CEW was prepared by carefully separating the white portion of the chicken egg from the whole egg followed by freeze-drying; the obtained solid dry white powder was used as such without any further purification. KAuCl4 (0.1 M, 0.5 mL) was added to a 10 mL aqueous solution containing CEW (25 mg/mL), under vigorous stirring, followed by the addition of NaOH solution (1 M, 2 mL) to bring the pH of the solution mixture to 10. The mixture was then incubated at 37 ◦ C for 6 h in dark without any disturbance. A schematic diagram of the procedure for the preparation of AuNCs stabilized by CEW under alkaline condition is shown in Scheme 1. 3. Results and discussion A number of experiments have been performed to optimize the conditions towards the formation of AuNCs with the maximum fluorescence emission. Factors such as the amount of the precursors, reaction temperature and time had to be considered. First, the amount ratio of CEW to KAuCl4 had to be fixed. A concentration

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Fig. 1. TEM images of gold nanoparticles formed (a) using 2.5 mg/mL of chicken egg white, (b) using ascorbic acid instead of sodium hydroxide, and (c) using optimised conditions.

Fig. 2. Time-dependent photoemission spectrum (ex = 535 nm) from 0 to 8 h for the reaction solution of KAuCl4 with CEW and sodium hydroxide at 37 ◦ C.

of 25 mg/mL of CEW was found to be the best and most suitable. Amounts lower than 25 mg/mL, i.e., 10 mg/mL resulted in AuNCs with very low fluorescence emission. When decreasing the concentration further to 2.5 mg/mL, gold nanoparticles sized ∼15 nm (Fig. 1a) were formed with no fluorescent emission. Increasing it above 25 mg/mL, a highly viscous solution was observed with very low fluorescence. In this study, we have used CEW under alkaline conditions for the synthesis of AuNCs. According to a previous report [25], alkaline condition was required for the reduction of the gold ions after its entrapment by the proteins present in CEW. The solubility of CEW in water was poor, resulting in a turbid solution, but after the addition of the NaOH solution, the turbidity disappears and the solution becomes clear by time. The turbidity was due to the poor insolubility of proteins such as ovotransferrin and ovomucoid, which on addition of NaOH dissolves in water thereby turning into a clear solution. Reactions carried out in the absence of NaOH for comparison led to the solidification of the mixture. Also, different ratios of KAuCl4 to NaOH were tried and finally a ratio of 1:40 was fixed, which gave the maximum fluorescence emission. In addition, studies were conducted replacing NaOH by ascorbic acid, which resulted in a wire-like network nanostructure (Fig. 1b). The

optimum temperature for the reaction had also to be determined. In this regard, the reactions were carried out at three different temperatures: room temperature (25 ◦ C), physiological temperature (37 ◦ C), and 74 ◦ C. We found that the maximum fluorescence was observed for experiments carried out at 37 ◦ C. After fixing the ratio between the precursors and the temperature, time-dependent studies on the fluorescence were carried. We found that the AuNCs with maximum fluorescent emission was observed in 6 h (Fig. 2) as the reaction finished in 6 h. Fig. 3a and b shows the UV–vis and fluorescence spectra of the reactions carried out at different conditions respectively. The AuNCs excited at 535 nm exhibited a maximum emission around 720 nm (Fig. 3b) emitting an intense red fluorescence under UV light (366 nm) and a weak absorption around 520 nm (Fig. 3a). Photographs showing the solid and the colloidal samples under visible and UV light are depicted in the Fig. 4a and b, respectively. Fig. 1c shows the TEM images of the AuNCs with an average size of 2 nm obtained using the optimized conditions. Studies on the chemistry of CEW were carried out to understand its role for the preparation of AuNCs. CEW is relatively homogeneous, containing very little particulate material and most of the solutes are proteins. This together with its ready availability has made CEW proteins some of the most studied by biochemists. Out of 10.5% proteins present in the liquid state, the main constituent proteins are ovalbumin 54%, ovotransferrin 12%, ovomucoid 11%, lysozyme 3.4%, and the rest are other proteins [27]. Table 1 summarizes the basic physical properties of the four major proteins in CEW proteins, with the different molar masses and isoelectric points. Ovalbumin, the chief protein in CEW is suspected to be the responsible for the formation of AuNCs. Thus, the protein ovalbumin was investigated separately and, indeed, it showed a similar behavior. This method was quite straightforward; we had to reduce the amount of NaOH added, maintaining the protein and the gold salt concentration and the temperature to acquire maximum fluorescence emission. The AuNCs prepared with OVA showed better fluorescence emission compared to the AuNCs obtained using CEW. Fig. 5a and b shows the comparative UV–vis and fluorescence spectra of AuNCs prepared using OVA and CEW, respectively. A time-dependent photo emission studies carried out using the

Fig. 3. (a) UV-vis and (b) fluorescence spectra (ex = 535 nm) for AuNCs at (i) 37 ◦ C, (ii) room temperature, (iii) 74 ◦ C, (iv) AuNPs formed using 2.5 mg/mL of CEW, and (v) AuNPs formed using ascorbic acid instead of NaOH.

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Table 1 Physical properties of the four major egg white proteins [27]. Proteins

Protein content (%)

Molar mass (kg/mol)

Carbohydrate content (%)

Isoelectric point

Type

Typical function

Solubility in water

Ovalbumin Ovotransferrin Ovomucoid Lysozyme

54 12 11 3.4

45.0 76.6 28.0 14.3

3.05 2.6 16.5–32.6 0

4.5 6.06 4.1 10.7

Glycoprotein Glycoprotein Proteinase inhibitors Bacteriolytic enzymes

Provide nutrition to yolk Iron transport Proteinase inhibitory function Protection from invading from bacteria

Very high – – Very high

Fig. 4. Photographs of chicken egg white (1) freeze dried powder, (2) aqueous solution,(3) aqueous dispersion, and (4) freeze-dried powder of CEW-AuNCs. (a) under visible light and (b) under UV light.

OVA showed a similar behavior in which a maximum emission was obtained in 6 h after which there was no change in the fluorescence intensity. Based on the fact that CEW is a multi-component system, it is difficult to predict the exact mechanism involved in the formation of AuNCs. However, recent reports have shown that aspartic acid, cysteine, glutamic acid, tyrosine, and tryptophan residues can reduce Au(III) or Ag(I) ions [30–32]. In the present study, it appears that the tyrosine and tryptophan residues present in the proteins of CEW is predominantly responsible for the reduction of gold ions. That is ascertained in the following way: tyrosine has been reported to reduce Au(III) or Ag(I) ions through their phenol group and their reduction capability can be strongly improved by adjusting the reaction pH above the pKa of tyrosine (∼10) [31]. Tryptophan has been reported to reduce metal ions in alkaline medium, when the indole NH group is deprotonated, which subsequently donates an electron to the nearby metal ion, forming a neutral tryptophyl radical. The formed radical subsequently reverses back to its native structure, converting to the kynurenine form of the peptide, or dimerizing to a ditryptophan peptide [32]. The addition of NaOH was very critical for the preparation of AuNCs, reactions carried out in the absence of NaOH resulted in the solidification of the CEW and KAuCl4 mixture after 6 h. Whereas, when ascorbic acid was replaced with NaOH, wire-like network gold nanostructure (Fig. 1b) was formed with no fluorescent emission but a broad surface plasmon resonance (Fig. 3). Thus, an alkaline condition is required to activate the phenol and indole group in the amino acid residues, tyrosine and tryptophan, respectively, which in turn reduces the Au3+ ions to Au0 . We speculated that the vibrational motion of the protein during the reaction at 37 ◦ C in alkaline condition might be responsible for the formation of AuNCs with a fluorescence emission and not

gold nanoparticles with a strong surface plasmon resonance band. Hence we carried out FTIR studies on CEW under four different condtions: native CEW, AuNCs formed with CEW, CEW with only NaOH, and KAuCl4 . The amide group vibrations of the backbone receive the most attention in protein IR spectroscopy because they are native to all proteins and report on secondary conformation and solvation. These include amide I (primarily CO stretch), amide II (CN stretch and NH in-plane bend), amide III (CN stretch, NH bend, and CO in-plane bend), and amide A (NH stretch) [33]. The FTIR spectra of the pure CEW and the AuNCs-CEW complex pellets were measured and the results are shown in Fig. 6a. The band corresponding to amide-I at 1653 cm−1 is intact. On the other hand, the presence of amide-II band at 1540 cm−1 and amide-III band at 1245 cm−1 was clearer in the pure CEW, while it was not so in the case of the composite. There exists an extensive overlapping of the amide-III band with in-plane deformational modes of O H bond (1450–1250 cm−1 ) as well as C O vibrations of esters (1330–1050 cm−1 ) and of aromatic anhydrides (1282–1220 cm−1 ). A very broad band of NH-stretch was also observed in the region 3000–3500 cm−1 . The FTIR spectra of the CEW–KAuCl4 and CEW–NaOH mixtures were also studied to understand the effect of NaOH in the reaction (Fig. 6b). In the case of CEW–KAuCl4 the amide I, II, and III are more prominent, whereas in the case of CEW–NaOH these bands tend to become weak, showing that there is free vibrational motion in CEW–KAuCl4 , but in the case of CEW–NaOH the vibrational modes are restricted to some extent by NaOH. A similar behavior was observed when the FTIR studies were carried with OVA and OVA-AuNCs. The exact mechanism involved is not clear; though a change in the amide bands are observed on the addition of NaOH. Thus, from the above observations we can speculate that, when the KAuCl4 is added to the CEW solution, the Au ions interact freely with the protein molecules primarily with the tyrosine and tryptophan residues. On the addition of NaOH the Au ions are reduced by the amino acid residues capable of reducing Au ions and are then capped by the proteins present in CEW. These quasi-encapsulated AuNCs do not grow in size because of the limited vibrational motion of the protein molecules by NaOH and the bulkiness of the proteins. Finally, the reproducibility of the experimental data was studied by carrying out ten replicate experiments using the optimized synthetic procedure. The obtained products were then compared using three different

Fig. 5. (a) UV–vis and (b) Fluorescence spectra (ex = 470 nm) for (i) AuNCs prepared with OVA and (ii) AuNCs prepared with CEW at 37 ◦ C.

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Fig. 6. FTIR spectra of (a) the pure chicken egg white (dotted), the AuNCs (solid) and (b) CEW–NaOH (dotted), CEW–KAuCl4 (solid) mixture.

bulkiness of the protein molecules restrict the growth of the AuNCs. We propose that the ovalbumin present in the CEW was predominantly responsible for the formation of AuNCs. The AuNCs thus prepared are highly stable both in the liquid and the solid form. Acknowledgment This work was supported by the WCU program through a grant provided by the Ministry of Education, Science and Technology (MEST) of Korea (Project No. R31-10026). References

Fig. 7. Plot representing the reproducibility of the experimental data as error bars characterized using three different characterization parameters.

characterization parameters and the deviation in the data was calculated and plotted as error bars in Fig. 7. The florescence intensity at 720 nm, size and the stability of the obtained AuNCs were used as the characterization parameters to study the reproducibility of the experiments. The AuNCs excited at 535 nm exhibited a maximum emission around 720 nm (Fig. 3b), hence the fluorescence intensities at 720 nm for the ten replicates were obtained and their standard deviation calculated. Similarly the standard deviations in the size of the AuNCs were calculated. To obtain the stability of AuNCs, the fluorescence intensities were studied for the replicates after a month and the deviation in the data calculated. The plot in Fig. 7 shows that all the three characterization parameters have a very low standard deviation which directly correlates with the reproducibility of the experimental data. 4. Conclusion For the first time chicken egg white has been exploited towards the synthesis of gold nanoclusters. We have succeeded in developing a simple, rapid, straightforward, low-cost, and green method for preparing highly fluorescent, water-soluble, and few atomcontaining gold nanoclusters at physiological temperature (37 ◦ C) using CEW. The experimental conditions have been standardized to obtain AuNCs with high fluorescence emission. The sodium hydroxide along with the proteins plays an important role in the reduction of the gold ions and subsequently the stabilization of AuNCs at 37 ◦ C. Prior to the reduction, the gold ions interact strongly with the amino acid residues. The addition of NaOH then results in the reduction of Au ions. It is speculated that tyrosine and tryptophan are the main amino acids responsible for the reduction of gold ions under alkaline conditions. The limited vibrational motion and the

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